Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging

Abstract

PurposeHuman epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed ankyrin repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application.MethodsG3 DARPins tagged with hexahistidine (His6) or with histidine glutamate (HE)3 and untagged G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with 125I, or with 111In via DOTA linked to a C-terminal cysteine. BALB/c mice were injected with radiolabelled G3 and tissue biodistribution was evaluated by gamma counting. The lead construct ((HE)3-G3) was assessed in mice bearing HER2-positive human breast tumour (BT474) xenografts.ResultsFor both isotopes, (HE)3-G3 had significantly lower liver uptake than His6-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His6-G3 and untagged G3. (HE)3-G3 was taken forward for evaluation in mice bearing HER2-positive tumour xenografts. The results demonstrated that radioactivity from 111In-(HE)3-G3 was better maintained in tumours and cleared faster from serum than radioactivity from 125I-(HE)3-G3, achieving superior tumour-to-blood ratios (343.7 ± 161.3 vs. 22.0 ± 11.3 at 24 h, respectively). On microSPECT/CT, 111In-labelled and 125I-labelled (HE)3-G3 could image HER2-positive tumours at 4 h after administration, but there was less normal tissue uptake of radioactivity with 111In-(HE)3-G3. Preadministration of trastuzumab did not affect the uptake of (HE)3-G3 by HER2-positive tumours.ConclusionRadiolabelled DARPin (HE)3-G3 is a versatile radioligand with potential to allow the acquisition of whole-body HER2 scans on the day of administration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00259-014-2940-2) contains supplementary material, which is available to authorized users.

Same-Day Imaging Using Small Proteins: Clinical Experience and Translational Prospects in Oncology

“…Also, several emerging sdAb-and scaffold protein-based probes that target other clinically relevant targets have been developed preclinically, and their clinical translation may impact future patient care. (35,36,38,40 …”

“…High tumor uptake was measured for both radionuclides in mice bearing human BT474 HER2-positive xenografts at 4 h after injection. However, the lower uptake of 111 In-compound in normal tissues led to higher tumor-to-background ratios than for its 125 I counterpart (36).…”

Dual-Purpose Linker for Alpha Helix Stabilization and Imaging Agent Conjugation to Glucagon-Like Peptide-1 Receptor Ligands

“…Low molecular weight agents were all filtered rapidly by the kidneys, but the smaller agents extravasated into the tumor more quickly. This has also driven protein engineers to find smaller scaffolds for rapid targeting 42–44 . It remains to be seen whether the same quantitative conclusions apply to the pancreas.…”

Designing repeat proteins for biosensors and medical imaging

“…DARPin G3 designed to specifically bind human epidermal growth factor receptor-2 (HER2) was used to image HER2 overexpression on breast cancer cells in vivo via selective attachment of 111 In-DOTA to a C-terminal cysteine residue ( Figure 2A) [25]. This labelling strategy is generally applicable to other repeat-protein scaffolds and thus holds promise for future clinical use.…”